Sensorineural hearing loss and vestibular disorders are permanent impairments for many people. They often stem from the loss of sensory hair cells in the inner ear, a population of cells that cannot be replaced when they die. Recent reports have shown that inner ear supporting cells, which neighbor sensory hair cells, can be coaxed into directly transdifferentiating into sensory hair cells in injured tissue. However, to achieve tue regeneration, inner ear supporting cells must also be guided into proliferation. We hypothesize that signaling through the ErbB2 receptor is sufficient to drive inner ear supporting cells to proliferate. We will test this hypothesis by over-expressing a constitutively activated (CA) ErbB2 receptor in auditory and balance supporting cells in the inner ear, and assessing subsequent proliferation. We present preliminary data with two independent CA-ErbB2 expression systems. Moreover, we show that both neonatal cochlear supporting cells and adult utricular supporting cells proliferate in response to CA-ErbB2 signaling in organ culture. Our first system uses two custom-built adenoviruses to over-express two mutated versions of the ErbB2 protein: one that is constitutively active, and one that is inert. In Aim 1, we will infect neonatal cochlear supportng cells with these viruses in vitro. We show preliminary data demonstrating that only the CA-ErbB2 virus activates phosphoinositol 3-kinase's (PI3K) regulatory subunit. Moreover, we show that the CA-ErbB2 virus drives proliferation among neonatal cochlear supporting cells in vitro. Surprisingly, CA-ErbB2 exerts some of its effects indirectly, as cells neighboring infected cells are also stimulated to divide. This finding highlights the power of using overexpression to investigate receptor function. We will quantify these results and probe the downstream effectors of the CA-ErbB2 receptor with reporter assays and chemical inhibitors. These experiments will define ErbB2's role in proliferation in neonatal mouse cochlear supporting cells. Our second system uses transgenic Tet-On technology to over-express a mutated, constitutively active ErbB2 protein in mouse supporting cells at different stages. We show preliminary data demonstrating that this system also activates PI3K. We also show that transgenic CA-ErbB2 signaling drives proliferation in neonatal cochlear supporting cells in vitro. Because Tet-On technology allows us to control both the timing and duration of the CA-ErbB2 signal, we will deliver a pulse of signaling and assess first if supporting cells divide, and second, if they trans differentiate into hair cells. In Aim 2, we propose to use this system to activate CA-ErbB2 signaling in cochlear supporting cells at different stages, with and without hair cell injury, to determine if ErbB2 signaling is sufficient to promote proliferation in vivo. In Aim 3, we will use both the CA-ErbB2 virus and the transgenic system to drive proliferation in adult utricular supporting cells in vitro, after hair cell injury. These experiments will illuminate how proliferaton in mammalian cochlear supporting cells can be regulated and significantly advance our understanding of inner ear regeneration.